Increase In Translational Activity Research Articles

Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation.

The mechanisms that govern organelle adaptation and remodelling remain poorly defined. The endo-lysosomal system degrades cargo from various routes, including endocytosis, phagocytosis, and autophagy. For phagocytes, endosomes and lysosomes (endo-lysosomes) are kingpin organelles because they are essential to kill pathogens and process and present antigens. During phagocyte activation, endo-lysosomes undergo a morphological transformation, going from a collection of dozens of globular structures to a tubular network in a process that requires the phosphatidylinositol-3-kinase-AKT-mechanistic target of rapamycin (mTOR) signalling pathway. Here, we show that the endo-lysosomal system undergoes an expansion in volume and holding capacity during phagocyte activation within 2 h of lipopolysaccharides (LPS) stimulation. Endo-lysosomal expansion was paralleled by an increase in lysosomal protein levels, but this was unexpectedly largely independent of the transcription factor EB (TFEB) and transcription factor E3 (TFE3), which are known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of acute organelle expansion via mTOR Complex 1 (mTORC1)-dependent increase in translation, which appears to be mediated by both S6Ks and 4E-BPs. Moreover, we show that stimulation of RAW 264.7 macrophage cell line with LPS alters translation of a subset but not all of mRNAs encoding endo-lysosomal proteins, thereby suggesting that endo-lysosome expansion is accompanied by functional remodelling. Importantly, mTORC1-dependent increase in translation activity was necessary for efficient and rapid antigen presentation by dendritic cells. Collectively, we identified a previously unknown and functionally relevant mechanism for endo-lysosome expansion that relies on mTORC1-dependent translation to stimulate endo-lysosome biogenesis in response to an infection signal.

Early Modern Translingualisms

The Renaissance period was marked by an increase in translation activity due to the invention of the moving printing press. However, the activity of translating was rather far from translation as we understand it in the modern sense and was often close to adaptation or even rewriting. Furthermore, translators sometimes produced a “translingual” text. This paper explores a corpus of Spanish texts translated into French during the 16th century. It analyses their translingual component and establishes a typology of translingualisms. It shows in particular that translingual writing was closely linked to the context of rivalries between languages and peoples.

Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling

The mammalian target of rapamycin (mTOR) pathway plays essential roles in the regulation of translational activity in many eukaryotes. Thus, from a bioprocessing point of view, understanding its molecular mechanisms may provide potential avenues for improving cell culture performance. Toward this end, the mTOR pathway of CHO cells in batch cultures was subjected to rapamycin treatment (inhibition) or nutrient supplementation (induction) and translational activities of CHO cells producing a monoclonal antibody (mAb) were evaluated with polysome profiling technology. Expectedly, rapamycin induced a shift of mRNAs from polysomes towards monosomes, thus reducing maximum cellular growth rate by 30%, while feeding additional nutrients extended mTOR pathway activity during the stationary growth phase in control batch culture, thereby contributing to an increase in global translation activity by up to 2-fold, and up to 5-fold higher specific translation of the heavy and light chains of the recombinant mAb. These increases in translation activity correlated with a 5-day extension in cellular growth and a 4-fold higher final product titer observed upon nutrient feeding. This first study of the relationship between the mTOR pathway and translational activity in CHO cultures provides key insights into the role of translational control in supporting greater productivity, which will lead to further enhancement of CHO cultures.

NAD+‐dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of mitochondrial ribosomal protein L10

Reversible acetylation regulates many biological processes including mitochondrial energy metabolism, cell survival and longevity. A member of the sirtuin family of NAD+‐dependent deacetylases, SIRT3, is important for regulation of reversible acetylation in mammalian mitochondria. In this study, we identified ribosomal MRPL10 as one of the major acetylated proteins in the mitochondrial ribosome. Ribosome associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+‐dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site‐directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 lead to an increase in translational activity of mitochondrial ribosomes in SIRT3−/− mice. In a similar manner, ectopic expression and knock‐down of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+‐ dependent deacetylase, SIRT3.Supported by NIH R01s to ECK (GM071034), YB (AG025223), and QT (DK075978).

NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10

A member of the sirtuin family of NAD(+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD(+)-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3(-/-) mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD(+)-dependent deacetylase, SIRT3.

Changes in histone modification upon activation of dormant mouse blastocysts

ABSTRACTGene expression in the implanting blastocyst is altered by stimulation with estrogen secreted from maternal ovaries. In the present study, to understand the mechanism regulating the changes in gene expression, diverse histone modifications in blastocysts were studied using a delayed implantation model, in which embryos were kept in a dormant state in the uterus by maternal ovariectomy and progesterone treatment, and then activated by injection with estrogen. Total transcriptional activity increased markedly in activated embryos, and immunocytochemistry with antibodies recognizing specific histone modifications revealed differential modification of several histones in the trophectoderm (TE) and inner cell mass (ICM) of dormant and activated embryos. High levels of histone H3 lysine 9 (H3K9) dimethylation, which suppresses gene expression, were observed in the ICM, but not in the TE, of dormant embryos, and the levels decreased when the embryos were activated, consistent with changes in transcriptional activity. Substitution of histone H3.3, a variant of H3, for dominant H3.1 increased in activated embryos, suggesting that histone substitution is involved in inducing gene expression associated with activation. In the nucleus, H3.3 was mainly localized in the nucleoli of activated embryos but not in those of dormant ones. In contrast, there were no obvious differences in the trimethylation of histone H3K9 or the acetylation of histones H3K9, H3K18 and H4K12 between dormant and activated embryos. These results suggest that a decrease in H3K9 dimethylation contributes to the acquisition of implantation competence by releasing genes from suppression. In addition, histone H3.3 substitution seems to be involved in global gene activation and facilitates the prompt recovery of dormant blastocysts to the active state by inducing rRNA synthesis, resulting in an increase in translational activity.

A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells

This review focuses on the striking differences in the patterns of transcription and translation in somatic and spermatogenic cells in mammals. In early haploid cells, mRNA translation evidently functions to restrict the synthesis of certain proteins, notably protamines, to transcriptionally inert late haploid cells. However, this does not explain why a substantial proportion of virtually all mRNA species are sequestered in translationally inactive free-messenger ribonucleoprotein particles (free-mRNPs) in meiotic cells, since most mRNAs undergo little or no increase in translational activity in transcriptionally active early haploid cells. In addition, most mRNAs in meiotic cells appear to be overexpressed because they are never fully loaded on polysomes and the levels of the corresponding protein are often much lower than the mRNA and are sometimes undetectable. A large number of genes are expressed at grossly higher levels in meiotic and/or early haploid spermatogenic cells than in somatic cells, yet they too are translated inefficiently. Many genes utilize alternative promoters in somatic and spermatogenic cells. Some of the resulting spermatogenic cell-altered transcripts (SCATs) encode proteins with novel functions, while others contain features in their 5'-UTRs, secondary structure or upstream reading frames, that are predicted to inhibit translation. This review proposes that the transcriptional machinery is modified to provide access to specific DNA sequences during meiosis, which leads to mRNA overexpression and creates a need for translational fine-tuning to prevent deleterious consequences of overproducing proteins.

Modifications of the 5' cap of mRNAs during Xenopus oocyte maturation: independence from changes in poly(A) length and impact on translation.

The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3' end of the mRNA can affect the state of the 5' cap structure, m7G(5')ppp(5')G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5' termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.

Mechanical wounding of potato tubers induces replication of potato virus S

The replication of potato virus S in potato tuber tissue was induced by wounding. An acidic 32kDa polypeptide accumulated in the polysome fraction of tubers upon wounding, concomitant with a transient increase in translational activity which is an integral component of the tuber wound response. We isolated and identified this polypeptide as the potato virus S coat protein. Immunoblot analyses indicated that the coat protein increased 2- to 20-fold in tubers within 4h of wounding. Northern blot analyses showed a parallel increase in potato virus S genomic RNA during the same time frame. Both potato virus S RNA and coat protein were barely detectable in tubers before wounding. By contrast, the significant amounts of coat protein detected in leaf and stem did not increase further upon wounding or treatment with abscisic acid or methyl jasmonate. The 32kDa polypeptide was not detected in seed tubers certified as virus-free. The observation that potato virus X and potato virus Y were also induced 5- to 10-fold within 4h of wounding led us to conclude that the induction of potato virus S in tubers resulted from the transient release from dormancy and general stimulation of tuber metabolism that occurs upon wounding. Wound-induction of potato virus S, potato virus X and potato virus Y may have implications on potato field practices or provide greater sensitivity in immunological screening of seed tubers for viruses.

Partial characterization of a unique 84-kDa polypeptide stimulated by ascorbic acid in skin fibroblasts.

Ascorbic acid stimulated the synthesis of a noncollagenous 84 kDa polypeptide by up to 3.5-fold in primary cultures of chick skin fibroblasts. Stimulation was observed with concentrations ranging 0.4 to 50 micrograms/ml and was dose-dependent within the concentrations up to 10 micrograms/ml during 24 h treatment under conditions in which procollagen synthesis was increased. This protein was found to be translated with a molecular size of 84 kDa in an in vitro cell-free translation experiment, indicating that the 84 kDa polypeptide was a primary translate. The stimulation was accompanied by a 1.7-fold increase in translational activity of the mRNA for this polypeptide. Its synthesis rapidly reduced when the cells were serially passaged until the third subcultivation, whereas procollagen synthesis remained essentially constant. These results indicate that the stimulation of this unique 84 kDa polypeptide by ascorbic acid was modulated at least in part at a translational level and its synthesis appears to be related to in vitro cellular aging.

Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers.

Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. The response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit [alpha] (EF-1[alpha]) parallels translational activity. Changes in the steady-state level of EF-1[alpha] mRNA, and expression of a chimeric EF-1[alpha] promoter/[beta]-glucuronidase construct in transgenic potato tubers, indicate that the gene encoding EF-1[alpha] is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors.

Ribosomal protein limitations in Escherichia coli under conditions of high translational activity

Details of the mechanism for ribosome synthesis have been incorporated in the single-cell Escherichia coli model, which enable us to predict the amount of protein synthesizing machinery under different environmental conditions. The predictions agree quite well with available experimental data. The model predicts that ribosomal protein limitations are important when the translational apparatus is in high demand. Ribosomal RNA synthesis is induced by an increase in translational activity, which, in turn, stimulates ribosomal protein synthesis. However, as the demand increases still more, the ribosomal protein mRNA must compete with the plasmid mRNA for ribosomes, and the efficiency of translation of ribosomal proteins is reduced. (c) 1994 John Wiley & Sons, Inc.

Regulation of eukaryotic translation initiation factor expression during T-cell activation

Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. Upon mitogenic stimulation the rate of protein synthesis increases 10-fold. We have studied the role of eIF-2 and eIF-4α (eIF-4E) expression in the mechanism of translational activation. During this period, the levels of eIF-2α and eIF-4α mRNA increase some 50-fold. Similar to the increase in ribosomes and mRNA, the number of eIF-2α, eIF-2β, and eIF-4α molecules per cell also increase 2–3-fold. This suggests that in addition to an increase in the pool size of translational components, an additional mechanism exists which results in an increased efficiency of factor utilization. We have looked at initiation factor phosphorylation. We find that eIF-2α does not undergo significant changes in its phosphorylation state nor is there a change in the efficiency of eIF-2 utilization. However, there is a rapid increase in the phosphorylation state of eIF-4α which correlates with the rapid increase in translational activity. It thus appears there are 2 distinct components responsible for the translational activation of quiescent T-cells during mitogenic stimulation. The first is the phosphorylation of eIF-4α, with a concomitant increase in the efficiency of eIF-4α utilization. The second is an increase in the pool sizes of eIF-2 and eIF-4α.

Maturation hormone induced an increase in the translational activity of starfish oocytes coincident with the phosphorylation of the mRNA cap binding protein, eIF-4E, and the activation of several kinases.

The stimulation of translation in starfish oocytes by the maturation hormone, 1-methyladenine (1-MA), requires the activation or mobilization of both initiation factors and mRNAs [Xu and Hille, Cell Regul. 1:1057, 1990]. We identify here the translational initiation complex, eIF-4F, and the guanine nucleotide exchange factor for eIF-2, eIF-2B, as the rate controlling components of protein synthesis in immature oocytes of the starfish, Pisaster orchraceus. Increased phosphorylation of eIF-4E, the cap binding subunit of the eIF-4F complex, is coincident with the initial increase in translational activity during maturation of these oocytes. Significantly, protein kinase C activity increased during oocyte maturation in parallel with the increase in eIF-4E phosphorylation and protein synthesis. An increase in the activities of cdc2 kinase and mitogen-activated myelin basic protein kinase (MBP kinase) similarly coincide with the increase in eIF-4E phosphorylation. However, neither cdc2 kinase nor MBP kinase phosphorylates eIF-4E in vitro. Casein kinase II activity does not change during oocyte maturation, and therefore, cannot be responsible for the activation of translation. Treatment of oocytes with phorbol 12-myristate 13-acetate, an activator of protein kinase C, for 30 min prior to the addition of 1-MA resulted in the inhibition of 1-MA-induced phosphorylation of eIF-4E, translational activation, and germinal vesicle breakdown. Therefore, protein kinase C may phosphorylate eIF-4E, after very early events of maturation. Another possibility is that eIF-4E is phosphorylated by an unknown kinase that is activated by the cascade of reactions stimulated by 1-MA. In conclusion, our results suggest a role for the phosphorylation of eIF-4E in the activation of translation during maturation, similar to translational regulation during the stimulation of growth in mammalian cells.

Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation.

The influence of extracellular matrix configuration on the tissue-specific function of cultured hepatocytes was investigated. Adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single layer of collagen gel for differences in the total RNA content, the level of albumin-specific mRNA, the rate of albumin gene transcription, and the rate of albumin mRNA translation. Adult hepatocytes in the sandwich system maintained the level of albumin mRNA similar to that found in the normal liver for at least six weeks, whereas the level of albumin mRNA declined rapidly in the single gel system. After one week of culture, hepatocytes in the single gel system could be induced to recover the high level of albumin mRNA and albumin production when a second layer of collagen gel was overlaid at that time. Furthermore, sandwiched hepatocytes maintained significantly higher transcriptional activity compared to cells in the single gel system. In addition to transcriptional control, the ultimate rate of albumin production was shown to depend on the rate of translation, which increased with culture time and reached a plateau in one to two weeks. This increase in translational activity over time in culture was observed in both the sandwich and the single gel systems and, thus, appeared to be independent of the configuration of extracellular matrix.

Light effects on DNA and protein synthesis in generative and non-generative cells of the antheridium of Chara vulgaris (Charophyta)

Studies of the effects of light on antheridial cells in Chara vulgaris Linnaeus (Charophyta) show that changes in the dynamics of antheridial filament growth are closely correlated with metabolic activity in manubria and capitular cells. Inhibition of DNA synthesis in darkness can be observed in antheridial filaments during mitotic divisions and during endoreplication in capitular cells and manubria. During spermatogenesis, when the number of cells in the antheridial filaments increases, a decrease in sensitivity to the absence of light in all antheridium cells is observed. The least sensitive period to darkness occurs at the time of differentiation of spermatozoids (spermiogenesis). After plants have been transferred from light to darkness and from darkness to light, manubria, capitular cells and antheridial filaments in young antheridia respond by a considerable decrease or increase in translation activity, respectively. However, immediately after changing the light conditions, the strongest response is found in manubria, being weaker in capitular cells and weakest in antheridial filaments.

Preliminary characterization of molecules that increase cell free translational activity of cardiac cytoplasmic RNA.

Previous evidence indicates that cells generate molecules that alter transcription in response to stress. Accordingly, we conducted preliminary experiments to characterize these molecules. Extracts were prepared from normal and experimental canine hearts in which a 100 mmHg gradient was created across the ascending aorta for 6 h. Experimental extracts were then treated by ultrafiltration through YM 10 and YM 30 membranes, by incubation with immobilized trypsin for 1 h at 37 degrees C and by incubation in a boiling water bath for 10 min. Extracts were perfused through isolated rat hearts for 1 h. Total cytoplasmic RNA was then extracted from the perfused heart and translated in a cell free medium containing [35S]-methionine. Incorporated label into newly synthesized protein was determined by liquid scintillation counting. A 13% mean increase in translational activity was produced by the fraction of the experimental extract passing through the YM 10 membrane compared with the material retained by YM 10 and YM 30 membranes. A 14% mean decrease in translational activity was observed in hearts perfused with experimental extracts treated with immobilized trypsin compared to untreated experimental extracts. There was no significant difference in translational activity of hearts perfused with experimental extracts subjected to boiling compared with non-boiled experimental extracts. These data suggest that the active molecules may be heat stable peptides or peptide containing substances of 10000 daltons or less in molecular weight.

Structural diversity and differential light control of mRNAs coding for angiosperm glyceraldehyde-3-phosphate dehydrogenases

Subunits A and B of chloroplast glyceraldehyde-3-phosphate dehydrogenase are synthesized as higher molecular weight precursors when polyadenylylated mRNA from angiosperm seedlings is translated in vitro by wheat germ ribosomes. The in vivo levels of mRNA coding for these precursors are strongly light dependent, and the increase in translational activity stimulated by continuous white light, relative to dark-grown seedlings, is at least 5- to 10-fold for the seven plant species investigated. As opposed to this, light does not seem to change mRNA levels coding for cytosolic glyceraldehyde-3-phosphate dehydrogenase, and the polypeptides synthesized in vitro have the same size as the authentic subunits. In addition, precursors of the chloroplast enzyme were identified for 12 different angiosperm species and compared with their respective subunits synthesized in vivo. The patterns of the in vitro and in vivo products correlate in several major characteristics. They both display a remarkable interspecific heterogeneity with respect to size and number of polypeptides. The peptide extensions of the enzyme precursors calculated from these data vary between 4,000 and 12,000 daltons and seem to fall into three major size classes. The present data demonstrate that chloroplast glyceraldehyde-3-phosphate dehydrogenase, like its cytosolic counterpart, is encoded in the nucleus. Yet, the two dehydrogenases are controlled differently at both the ontogenetic and phylogenetic levels. They follow separate biosynthetic pathways with respect to light regulation, post-translational processing, and transport and also exhibit different evolutionary rates. The fast evolutionary change observed for the chloroplast enzyme contrasts sharply with the conservative structure and sequence of the cytosolic enzyme.